There is a critical gap in our understanding of how a deadly fungal pathogen such as Cryptococcus neoformans invades the central nervous system (CNS). The continued existence of this gap has resulted in a major deficit in our grasp of fungal disease of the CNS and has led to a shortfall in the development of more effective and innovative treatment options. Current evidence on cerebral pathogenesis of C. neoformans has shown a predominantly transcellular migration of cryptococci across the brain endothelium (a.k.a. the blood- brain barrier); but the identities of brain endothelium factors and key virulenc fungal factors that promote this process are largely unknown. The objective in this particular application is to understand how a newly identified, secreted fungal metalloprotease targets brain endothelial proteins and how this association transforms the microenvironment within the fungal-brain endothelium interface to promote the migration of fungal cells into the CNS. The central hypothesis is that a newly identified secreted metalloprotease (Mpr1) stimulates the transmigration of cryptococci into the CNS by unmasking/stimulating surface receptors/ligands and/or adhesion proteins via its proteolytic activity. The formulation of this hypothesis is based on strong preliminary data produced from the laboratory of the applicant. The rationale for the proposed research is that understanding the molecular mechanisms that induce cerebral pathogenesis of C. neoformans will translate into the development of advanced antinfective drugs and vaccines that could be highly effective in the treatment of cryptococcal disease which claims the lives of ~600,00 individuals per year. Guided by strong preliminary data, this hypothesis will be tested by pursuing one specific aim: Resolve the role of the brain endothelium in the transmigration of C. neoformans into the CNS by identifying the host targets of Mpr1 that transform the surface microenvironment at the fungal-brain endothelium interface. Here we will test the prediction that Mpr1 directly targets surface proteins of the brain endothelium as a means to redefine the microenvironment of the fungal-brain endothelium to favor and promote fungal invasion of the CNS. We will do this by first establishing whether Mpr1 is sufficient for attachment to the brain endothelium by expressing Mpr1 in a non-pathogenic yeast and exploiting this phenotype to identify host targets of Mpr1. Secondly we will use a powerful, high-content proteomic screen to identify native host substrates of Mpr1. To prioritize and resolve the role of these targets, an in vitro model of the BBB, in vivo models of cryptococcal disease and real-time intravital microscopy will be utilized. The innovation of the proposed study lies in its use of an in vitro model of the BBB and state-of-the-art methodology to examine a novel mechanism utilized by C. neoformans to swarm the CNS. We anticipate that the proposed study will advance our understanding of the role of the brain endothelium in fungal disease of the CNS. Ultimately such knowledge has the potential to invigorate drug and vaccine development strategies for individuals suffering from fungal meningoencephalitis.